Cellular and molecular events controlling acquisition of cytotoxic activity by melanoma-reactive CD4⁺ T cells in vivo

While there is an abundance of studies on cytotoxic CD8⁺ T cells in cancer immunotherapy, CD4⁺ T cells with cytotoxic potential are receiving increasing attention from the scientific community. Previously, our lab has underscored the significance of tumour reactive CD4⁺ T cells which acquire cytotoxic activity during immunotherapy of malignant melanoma. This study aims to analyse and characterise the molecular and cellular mechanisms underlying the function of these cytotoxic CD4⁺ T cells. On protein and transcript level, cytotoxic tumour infiltrating CD4⁺Trp1 T cells exhibited a highly plastic phenotype: Th1 and Th2 specific transcription factors Gata3 and T-bet were co-expressed and inflammatory cytokines IFNγ, TNF-α and IL-2 were secreted. Additionally, CD8⁺ lineage specific transcription factor Runx3 expression was elevated and correlated highly with GzmB expression. However, and in contrast to classical CD8⁺ CTLs, cytotoxic CD4⁺Trp1 T cells lacked expression of CD8⁺ transcription factor Eomes. In depth microarray analysis via Canonical Correspondence Analysis (CCAM) revealed a high correlation of tumour infiltrating CD4⁺Trp1 cells with a full effector CD8⁺ T cell gene signature rather than a CD4⁺ or CD8⁺ memory phenotype. The strong correspondence with differentiated CD8⁺ effector T cells prompted the investigation of the role of mTOR signalling in CD4⁺ cytotoxicity as mTOR activity is crucial for CD8⁺ effector differentiation. Inhibition of mTORC1 activity by administration of rapamycin and genetic engineering of CD4⁺Trp1 cells was evaluated. Disruption of mTORC1 signalling counteracted the acquisition and/or maintenance of a cytotoxic phenotype whilst preserving the capacity to produce inflammatory cytokines. This study illustrates the complexity of this highly plastic, cytotoxic CD4⁺ T cell subset and highlights the importance of mTORC1 signalling for the cytotoxic activity of tumour specific CD4⁺Trp1 T cells